Acc 030

The control or NMD rats were deeply anesthetized with chloral hydrate and perfused transcardially with 0.9% isotonic saline followed by 4% paraformaldehyde (PFA) in saline. Then the brains were post fixed overnight in PFA. After that, the brains were transferred to a 20% sucrose solution followed by a 30% sucrose solution. For labeling, 12 μm sections of BLA were simultaneously incubated with synaptophysin (1:100, ab8049, Abcam) and TRPV1R (1:200, ACC-030, Alomone labs) antibodies for overnight at 4 °C and then incubated with secondary antibody with Alexa Fluor 488 and 594 for 2 hours at room temperature. Negative controls were performed without the primary antibody.

The lens was fixed in 0.75% PFA at room temperature for 12 h. Following fixation, all lenses were prepared for cryosectioning using established protocols [10 (link),13 (link)]. Cryosections were first treated with 0.1% Triton X-100 for 10 min at room temperature, followed by incubation in the blocking solution (3% bovine serum albumin and 3% normal goat serum in phosphate-buffered saline [PBS]) for 1 h. Sections were incubated in AQP0 antibody (1:100, B-11, Santa Cruz Biotechnology, Dallas, TX, USA), AQP5 antibody (1:100, AB15858, Millipore, Billerica, MA, USA), TRPV1 antibody (1:100, ACC-030, Alomone, Jerusalem, Israel), or TRPV4 antibody (1:100, ab39260, Abcam, Cambridge, UK) antibodies prepared in blocking solution at 4 °C. Slides were then incubated with goat anti-mouse or anti-rabbit Alexa Fluor 488 (1:200, A-11008, Thermo Fisher Science, Waltham, MA, USA) secondary antibody in blocking solution with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) to label the nuclei, followed by incubation with wheat germ agglutinin (WGA) Alexa Fluor 594 (1:100, W11262, Thermo Fisher Science) in PBS to label cell membranes. The coverslips were mounted using VectaShield HardSet™ anti-fade mounting medium (Vector Laboratories, Burlingame, CA, USA) and imaged using a laser scanning confocal microscope (Olympus FV1000, Tokyo, Japan).

Three polyclonal rabbit anti-TRPV1 antibodies [SC-20813, purified by proprietary techniques (Santa Cruz Biotechnology, CA, USA), ACC-030, affinity purified on immobilized antigen (Alomone Labs, Jerusalem, Israel), LS-C150735, affinity purified (Lifespan Biosciences, WA, USA), and polyclonal IgG goat anti-rabbit secondary antibody-FITC (Santa Cruz Biotechnology) were initially assessed. Testing of fixation and permeabilization kits [CALTAG™ (Life Technologies); Cytofix/Cytoperm™ (BD Biosciences)], blocking agents [AB serum (from a healthy donor), 10% goat serum (Life Technologies), FcR blocking reagent (Miltenyi Biotechnology, Cologne, Germany) and a mixture of 10% human AB serum, 1% BSA and 0.05% sodium azide, and goat serum mix (10% goat serum + 0.1% BSA + 0.05% sodium azide)] was also performed. Isotype control rabbit IgG, sc-3888 (Santa Cruz Biotechnology) was used as a negative control in a same quantity/dilution as the primary anti-TRPV1 in all experiments. Exclusion of TRPV1 antibody was used as an additional negative control. Protocols were developed to produce the best signal (specific binding) to noise (non-specific binding) ratio for Western blotting and separation of TRPV1 signal from isotype control for flow cytometry.

Equal protein concentrations of the cleared cell lysates were used for immunoprecipitation. Each sample was incubated overnight and end-to-end mixing with 10⁴ anti-ARMS (mouse, MA1-90667, Invitrogen, Darmstadt, Germany) coupled MagPlex Microspheres (Luminex Corp., Hertogenbosch, The Netherlands). Incubated MagPlex Microspheres were washed twice with PBST and incubated for 1 h at 900 rpm with anti-ARMS (rabbit, ab34790, abcam, Berlin, Germany), anti-TRPV1 (rabbit, ACC-030, Alomone Labs, Jerusalem, Israel), anti-AKAP79 (rabbit, D28G3, Cell Signaling, Leiden, The Netherlands), or anti-Phospho-PKA substrate (rabbit, 100G7E, Cell Signaling, Leiden, The Netherlands). After antibody incubation, MagPlex Microspheres were washed twice with PBST, incubated for 1 h at 900 rpm with anti-Rabbit-PE (goat, P-2771MP, Invitrogen, Darmstadt, Germany) and washed twice with PBST again. MagPlex Microspheres were resuspended in PBST and analyzed via FLEXMAP 3D (Luminex Corp., Hertogenbosch, The Netherlands).

Simultaneous detection of Ghrelin receptor and ASIC3 or TRPV1 was performed in retrolabeled NG. 10-μm frontal sections were preincubated for 1 h with 0.1 M PBS (pH 7.4) containing 0.3% Triton X-100, and 5% fetal bovine serum (FBS), then incubated overnight at 4 °C with mouse monoclonal anti-Ghrelin receptor antibody (1:200, SC-374515, Santa Cruz Biotechnology, Germany), and guinea pig polyclonal anti-ASIC3 antibody (1:250, AB5927, Millipore, USA) or rabbit polyclonal anti-TRPV1 antibody (1:200, ACC-030, Alomone Labs, Israel) diluted in the same buffer. After washing, the sections were incubated for 90 min with the secondary antibody conjugated either to Cy-3 or Alexa Fluor 488 (1:500; all secondary antibodies were purchased from Life Technologies). In all conditions, sections were finally coverslipped with mounting medium for fluorescence microscope preparation. Sections were observed with Zeiss Axio Imager Z1 microscope equipped with AxioCam Icc3 digital camera (Carl Zeiss, S.A., Barcelona, Spain). Appropriate positive controls were used to check antibody specificity.

Murine dorsal root ganglionic neurons (mDRGs) grown in 24 well plates were infected with lentiPirtLCA (5.4 × 10⁴ TU/well, ~5.4 MOI) for 5 days before fixation with 3.7% formaldehyde. After washing, specimens were permeabilized in phosphate-buffered saline with 0.1% Triton X-100 and then incubated in PBS containing 1% BSA at room temperature for 1 h. Samples were then incubated with rabbit polyclonal antibody to TRPV1 (1:100, Cat. No. ACC-030, Alomone, Shanghai, China) together with mouse monoclonal antibody to the LCA cleaved SNAP-25 (1:500, Cat. No. MBS350064, MyBioSource, sourced from the representative company Biolead, Beijing, China) in blocking solution (4 °C, overnight). The specimens were washed in PBS and incubated with donkey anti-mouse Alexa 488 and donkey anti-rabbit Alexa 594. After the final wash of the secondary antibody, specimens were incubated with prolong anti-fade reagents containing 4′, 6-Diamidino-2-phenylindole dihydrochloride. Images were taken using a IX73 Olympus inverted microscope using CellSens Dimension Imaging software.